IFN-? Combined With LPS Enhances The Effect Of Mesenchymal Stem Cells And Their Derived Extracellular Vesicles On Regulatory T Cells

Background and Objective: Chronic graft-versus-host disease(c GVHD)is a severe complication after allogeneic hematopoietic stem-cell transplantation.Mesenchymal stem cells(MSCs)have been showed to possess immunoregulatory functions,which makes MSCs alleviate chronic GVHD by upregulating the proportion of regulatory T cells.Later,it was reported that MSCs were plastic in their immunomodulatory abilities,mainly manifested as immune promotion and immunosuppression,which were related to the microenvironment and the TLR3 and TLR4 expression of MSCs.It also leads to the heterogeneity and instability in clinical application.Recently,it has been found that MSCs exert their functions mainly by extracellular vesicles.Mesenchymal stem cells-derived extracellular vesicles(MSC-EVs)have similar immunoregulatory functions to their mother cells,and the functions are not affected by the microenvironment due to the lack of cellular structure,which makes MSC-EVs more stable and safer.MSC-EVs may replace MSCs as a new cell-free therapy.Therefore,it is of great clinical significance to prepare MSCs and MSC-EVs with high efficiency and stable immunosuppressive functions.It was found that IFN-? combined with proinflammatory factors such as IL-1?,IL-1?,TNF-? could enhance the immunosuppression of MSCs.Moreover,the levels of IFN-? and LPS in serum of GVHD increase significantly,could it favor MSCs to exert immunosuppressive function? And what about IFN-? combined with LPS treated MSCEVs? This study will investigate the effect of IFN-? combined with LPS on the immunomodulation of MSCs and MSC-EVs in the inflammatory microenvironment induced by IFN-? and LPS in vitro,and further investigate the therapeutic effect of IFN-? combined with LPS treated MSC-EVs on GVHD in vivo.Methods: 1? Human umbilical cord derived mesenchymal stem cells(h UC-MSCs)were isolated from about 10 cm umbilical cord of full-term healthy newborns by type ? collagenase digestion and adherence method.And they were identified by morphological characteristics,surface markers and the abilities of differentiation at passage three.This study was approved by the medical ethics committee of Guangdong general hospital.Umbilical cord donors signed informed consent to donate.2? h UC-MSCs treated with IFN-? and LPS were cocultured with peripheral blood mononuclear cells from healthy volunteers under direct or indirect contact to analyze the proportion of regulatory T cells.The RNA of h UC-MSCs after coculture was extracted by Trizol method,and the m RNA expression levels of TLR3 and TLR4 in h UC-MSCs were analyzed by fluorescence quantitative PCR.3? MSC-EVs were harvested by ultracentrifugation and identified by transmission electron microscopy,Nanosight and Western blot.4? IFN-? combined with LPS treated MSC-EVs were cocultured with PBMC in vitro to analyze the proportion of regulatory T cells by flow cytometry.5? A humanized GVHD mouse model was established and randomly divided into three groups,including PBS control group,untreated MSC-EVs treatment group,and IFN-? combined with LPS treated MSC-EVs treatment group,to observe the clinical manifestations and survival,and analyze the proportion of regulatory T cells.Results: 1?h UC-MSCs were successfully isolated by type ? collagenase digestion and adherence method.They could adhere to plastic culture dishes and exhibited fibroblast-like or spindleshaped morphology.They highly expressed CD105,CD90 and CD73,and lacked expression of HLA-DR,CD45 and CD34.And they could also differentiate into osteoblasts or adipocytes.2?Whatever under direct contact or indirect contact,h UC-MSCs treated with IFN-? and LPS could increase the percentage of regulatory T cells,which was better than h UC-MSCs treated with/without IFN-? or LPS alone(the percentage of regulatory T cells of IFN-?+LPSMSCs group?un-MSCs group?IFN-?-MSCs group and LPS-MSCs group:day 5 under direct contact——12.8±0.8% vs 10.7±1.2% vs 11.5±0.7% vs 8.7±1.2%,P<0.05;day 3 under indirect contact——15.9±1.6% vs 10.4±0.8% vs 12.6±1.2% vs 11.8±1.3%,P<0.01).What's more,under indirect contact,h UC-MSCs treated with IFN-? and LPS could more quickly increase the percentage of regulatory T cells.Meanwhile,IFN-? combined with LPS could significantly upregulate the m RNA expression levels of TLR3 in h UC-MSCs(the m RNA expression levels of TLR3 in IFN-?+LPS-MSCs group?un-MSCs group?IFN-?- MSCs group and LPS-MSCs group:1.38±0.32 vs 1 vs 1.1±0.1 vs 0.76±0.15,P<0.05),and showed a strong trend of upregulation of the m RNA expression levels of TLR4(P=0.08),but had no significant difference on the ratio of TLR3 and TLR4(P>0.05).3?The MSC-EVs extracted by ultracentrifugation exhibited saucer-like morphology with a particle size range of 95.9 nm-201.7nm,an average of 146.4nm and a maximum peak of 108 nm,and expressed CD63,which was consistent with the characteristics of EVs.4?Compared with untreated MSC-EVs,IFN-? combined with LPS treated MSC-EVs could significantly increase the percentage of regulatory T cells when cocultured with PBMC(the percentage of regulatory T cells of IFN-?+LPS-EVs group?PBMC group and un-EVs group:day 3——27±1.2% vs 20.7±1.4% vs 22.6±1.6%,P<0.01;day 4——13.4±1.7% vs 7.7±2% vs 9.9±0.9%,P<0.01).5?In vivo,IFN-? combined with LPS treated MSC-EVs could increase the percentage of regulatory T cells,improved the clinical manifestation of GVHD,and prolonged the survival time(the percentage of regulatory T cells of PBS group?un-EVs group and IFN-?+LPS-EVs group:4±0.6% vs 10.9±1% vs 10.6±1.1%,P<0.05;the survival time:55.8±8.8 vs 66±10 vs 66.2±8.2,P<0.05),but there was no significant difference when compared with the untreated MSC-EVs(P>0.05).Conclusions: IFN-? combined with LPS treated MSC-EVs could upregulate the percentage of regulatory T cells in PBMC in vitro,which was superior to untreated MSC-EVs.What's more,IFN-? combined with LPS treated MSC-EVs could also upregulate the percentage of regulatory T cells,improve the clinical manifestation and prolong the survival time in GVHD.